Recently, much attention has focused on the therapeutic potential for the individual components of the fibrinolytic system and especially for the plasminogen activators because they can control the initiation of the process.
Thrombolytic therapy is usually carried out with urokinase, a serine protease isolated from human urine, or streptokinase, a bacterial protein. Both of these plasminogen activators can activate plasminogen to form plasmin and are employed in clinical practice today. However, urokinase has a very short (3-15 minutes) useful half-life following its injection into humans since it exists in an enzymatically fully active form which is inactivated by the relatively high concentrations of protease inhibitors in the body fluids. The short half-life for this material has required that large amounts of urokinase be infused during therapeutic treatment.
Since it is easier to obtain and far less expensive than urokinase, streptokinase is the most widely used thrombolytic agent at present. However, its use in thrombolytic therapy is limited due to its bacterial origin. Streptokinase is a plasminogen activator produced by Lancefield group C strains of beta-hemolytic streptococci. Unlike urokinase which directly activates plasminogen to plasmin, streptokinase activates the fibrinolytic system indirectly by complexing with plasminogen to produce an active modified plasminogen moiety. Thrombolysis with both of these substances, urokinase and streptokinase, however, is usually associated with systemic activation of plasminogen which can produce indiscriminate digestion of circulating coagulation proteins such as fibrinogen and alpha-2-antiplasmin, and significantly increase the risk of haemorrhage during treatment. Thus, these agents are said to lack thrombolytic selectivity or fibrin specificity in their activation of plasminogen.
Plasminogen activators have been extracted from normal and tumor tissues and are produced by certain cells in culture. The plasminogen activators derived from these sources are serine proteases with a high thrombolytic selectivity or fibrin specificity in their plasminogen activation. Thus, these activators convert plasminogen to plasmin in a selective manner, causing lysis of fibrin clots while preserving much of the circulating coagulation proteins. There are two types of plasminogen activators with high thrombolytic selectivity--a single-chain urokinase-type plasminogen activator (scu-PA) also known as prourokinase (PUK), and tissue-type plasminogen activator (TPA)--which are easily distinguished by differences in their immunological properties. The single-chain urokinase-type plasminogen activator (scu-PA) is a trypsin-like serine protease 411 amino acid residues in length. The molecule contains three domains: a cysteine-rich amino-terminal region of 45 amino acid residues which is homologous with epidermal growth factor (EGF) and thus is termed the EGF domain; a kringle region or kringle domain which comprises the 87 amino acid residues immediately adjacent to the EGF domain; and a serine protease region, with the active site residues histidine, aspartate and serine, in the carboxy-terminal region of the molecule.
Tissue type plasminogen activator (TPA) is also a serine protease and is composed of a single polypeptide chain of 527 amino acids. The primary structure of TPA shares a high degree of homology with that of scu-PA. For example, TPA is converted by plasmin cleavage of the arginine 275-isoleucine 276 bond to a two chain form also linked by at least one disulfide bond. However, unlike scu PA, TPA is not cleaved by thrombin (Ichinose et al., 1986. J. Biol. Chem. 261, 3486-3489).
Single chain uPA (scu-PA) has been shown to be an effective and fibrin selective plasminogen activator by measurements of preservation of the circulating coagulation proteins fibrinogen and alpha-2-antiplasmin during clot lysis in vivo in a rabbit model system (Stump et al., 1987, Blood 69: 592-596) and in a dog model system (Collen et al., 1985, Circulation 72: 384-388). By contrast plasmin-generated tcu-PA (cleaved at the lysine 158-isoleucine 159 peptide bond) is less effective in vivo and considerably less fibrin selective.
Hydrolysis of the lysine 158-isoleucine 159 bond by plasmin converts the single chain scu-PA into urokinase, a two chain scuPA (plasmin generated tcu-PA) in which the chains are linked to one another by at least one disulfide bridge. In addition, purified human thrombin recently has been shown to cleave the arginine 156 phenylalanine 157 bond to yield a similar but different two-chain uPA, a thrombin-generated tcu PA in which the chains are also held together by at least one disulfide bond (Ichinose et al., 1986. J. Biol. Chem. 261, 3486-3489). A preparation of enzymes from the venom of the snake Agkistrodon contortrix produces two chain u-PA with identical properties as measured in vitro (Gurewich and Pannell, 1987. Blood, 69, 769-772), and may result from cleavage of the same arginine 156 phenylalanine 157 bond.
Both cleavage by plasmin and cleavage by thrombin may play important physiological roles and have been demonstrated to produce molecules with quite different properties as measured in vitro. For example, plasmin cleavage produces a tcu-PA which is more active than scu-PA on small molecule substrates such as S2444 (N-pyro-Glu-Gly-p-nitroanilide), while thrombin cleavage produces a tcu-PA which is inactive on S2444 and which currently is considered to be non-cleavable by plasmin (Ichinose et al., 1986, supra; Gurewich and Pannell, 1987, supra). The properties of plasmin-produced tcu-PA (ie., urokinase) as measured in in vivo thrombolytic systems reveal it to be inferior to scu-PA with regard to efficiency of clot lysis (potency) and fibrin specificity (Stump et al., 1987, Blood 69: 592-596; Collen et al., 1985, Circulation 72: 384-388). In contrast, nothing is known about the properties of thrombin-generated tcu-PA in in vivo thrombolytic systems. Current theory holds that fibrin-specificity is a property of single-chain full length u-PA and single-chain truncated u-PA (scu-PA-32k) beginning with leucine residue 144 (Stump et al., 1987, supra). Consistent with this notion, plasmin generated tcu-PA (ie., urokinase) lacks fibrin-specificity in its initation of clot lysis in vitro and in vivo. At present, no two-chain urinary plasminogen activators are generally known to the field to have fibrin-specificity. Thrombin-generated tcu-PA has been considered inactive and unactivable; indeed, the generation of tcu PA cleaved by thrombin has been regarded as a normal physiological mechanism for preventing formation of plasmin-generated tcu-PA and thereby down-modulating the activity of scu-PA (Gurewich and Pannell, 1987, supra).
Unexpected new uses of the thrombin-generated tcu-PA molecule which permit use in therapeutic methods of treatment for pulmonary embolism, deep vein thrombosis, heart attack and stroke, have been discovered and are the subject of the present invention. These new uses include efficient clot lysis in vivo with a high degree of fibrin specificity. Thus, a plasminogen activator useful for thrombolysis has been made available as a new therapeutic agent.